Skin cancer is strongly associated with ultraviolet (UV) radiation that generates many mutations. There is thus a need to investigate what mutations drive skin cancer. Silencing of the CDKN2A gene, which encodes the p16 tumor suppressor protein, plays a key role in cancer progression. Our previous mouse study showed that chronic UV irradiation to skin induced mutations in the regulatory and gene body regions of tumor suppressor genes, including the Cdkn2a/p16 promoter. Importantly, topical application of caffeine to mouse back skin reduced the frequency of these Cdkn2a/p16 promoter mutations and suppressed skin cancer development. However, the impact of these promoter mutations on cancer progression remained unclear. We hypothesized that these mutations may inhibit the binding of critical transcription factors, thereby reducing the expression of the p16 tumor suppressor. The mutations at the Cdkn2a/p16 promoter were found in the DNA sequence that was similar to the ETS transcription factor-binding element (EBE). To determine the functionality of this DNA sequence at the Cdkn2a/p16 promoter (p16-EBE), we cloned this putative EBE into the reporter construct in which the binding of transcription factors to p16-EBE drives luciferase expression. ETS1 and ETS2 represent the ETS transcription factor family, with the latter expressed dominantly in skin. We found that overexpression of either mouse ETS1 or ETS2 resulted in higher luciferase activities than no overexpression control, indicating that ETS transcription factors bind to p16-EBE. We also tested mutated p16-EBE that carries the UV-induced mutation found in mice. With overexpression of mouse ETS1 or ETS2, the mutated p16-EBE showed markedly lower luciferase activities than wild-type counterpart, implying that the UV-induced mutation in p16-EBE inhibits the binding of ETS proteins to the Cdkn2a/p16 promoter. This study highlights the importance of loss-of-function mutations in promoters that may contribute to cancer progression.